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Biomineralization of Gold Nanoparticles by Lysozyme and Cytochrome c and Their Applications in Protein Film Formation )

Mandeep Singh Bakshi,*,† Harpreet Kaur,‡ Tarlok Singh Banipal,‡ Narpinder Singh,§ and Gurinder Kaur † Department of Chemistry, Mount Saint Vincent University, Halifax, Nova Scotia B3M 2J6, Canada, Department of Chemistry, and §Department of Food Science and Technology, Guru Nanak Dev University, Amritsar 143005, Punjab, India, and Nanotechnology Research Laboratory, College of North Atlantic, Labrador City, NL A2 V 2K7 Canada )

‡

Received April 28, 2010. Revised Manuscript Received June 29, 2010 Lysozyme (Lys) and cytochrome c (Cyc,c) proteins were used as mild reducing and stabilizing agents to synthesize gold nanoparticles (NPs) at precisely 40 and 80 °C. All reactions were monitored simultaneously by UV-visible measurements to determine changes in the nature of the protein during the course of reaction. The synthesis of Au NPs caused the simultaneous denaturation of protein due to the formation of bioconjugate NPs, and the denaturation temperature decreased with the number of NPs. Lys entrapped NPs in a typical gel state, and Cyc,c carried them on welldefined micelles at 80 °C or in the form of long fibrils or strands at 40 °C. The shape, size, and arrangement of bioconjugate NPs were characterized by atomic force microscopy and transmission electron microscopy measurements. Purified bioconjugate NPs were further used in zein protein film formation. The resulting films were characterized by photophysical and mechanical measurements. The induction of bioconjugate NPs made protein films isotropic and relatively more brittle (with a greater effect for Cyc,c than for Lys conjugate NPs) than in their absence and was considered to be well suited for biomedical applications.

Introduction 1

Biomineralization is a quickly developing area of materials science where biomolecules are directly involved in the synthesis of different materials. The inherent complexity of biomolecules makes even simple reactions difficult to understand. The conformational changes along with the folding and unfolding mechanisms of proteins significantly influence the overall reaction when a protein is directly involved in the synthesis of biomaterials.1h The use of simple, low-molecular-weight proteins such as lysozyme (Lys) and cytochrome c (Cyt,c) sometimes makes it easier to follow and understand their involvement in bioconjugate materials. In this work, we have focused our attention on the direct synthesis of such materials by using simple, well-studied proteins such as Lys and Cyc,c as weak reducing and capping/stabilizing agents1g-j and then use such bioconjugate materials in protein film formation. This helps us to understand the mechanism in which a simple protein is involved to produce metal nucleating centers that are subsequently capped and stabilized by the protein to generate bioconjugate nanomaterials.1h Here, the physical state of a protein is important in configuring the overall shape and structure of nanomaterials.1h,i It not only controls the nucleation process effectively but also produces welldefined bionanomaterials that are very useful in important *Corresponding author. E-mail: [email protected]. (1) (a) So, C. R.; Kulp, J. L.; Oren, E. E.; Zareie, H.; Tamerler, C.; Evans, J. S.; Sarikaya, M. ACS Nano 2009, 3, 1525. (b) Wang, X.; Muller, W. E. Trends Biotechnol. 2009, 27, 375. (c) Muthukumar, M. J. Chem. Phys. 2009, 130, 161101. (d) Villarreal-Ramirez, E.; Moreno, A.; Mas-Oliva, J.; Chavez-Pacheco, J. L.; Narayanan, A. S.; Gil-Chavarria, I.; Zeichner-David, M.; Arzate, H. Biochem. Biophys. Res. Commun. 2009, 384, 49. (e) Nuraje, N.; Mohammed, S.; Yang, L.; Matsui, H. Angew. Chem., Int. Ed. 2009, 48, 2546. (f) Kunz, W.; Kellermeier, M. Science 2009, 323, 344. (g) Xie, J.; Lee, J. Y.; Wang, D. I. C.; Ting, Y. P. ACS Nano 2007, 1, 429. (h) Bakshi, M. S.; Thakur, P.; Kaur, G.; Kaur, H.; Banipal, T. S.; Possmayer, F.; Petersen, N. O. Adv. Funct. Mater. 2009, 19, 1451. (i) Bakshi, M. S.; Jaswal, V. S.; Kaur, G.; Simpson, T. W.; Banipal, P. K.; Banipal, T. S.; Possmayer, F.; Petersen, N. O. J. Phys. Chem. C 2009, 113, 9121. (j) Kaur, G.; Iqbal, M.; Bakshi, M. S. J. Phys. Chem. C 2009, 113, 13670.

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biological applications such as protein film formation. The factors that significantly influence this process are the selfaggregation, folding, and unfolding of the protein tertiary structure.1h In addition, the predominant hydrophobic or hydrophilic nature of a protein is the main contributing factor in determining the final state of protein aggregates. Lys contains 129 amino acids arranged in a single polypeptide chain with an average formula weight of 14 300 and is freely available in plant and animal tissues. It is also abundant in egg whites, human tears,2 saliva,3 and endocrine glands4 and is highly surface-active.5 The surface activity is primarily related to its hydrophobic nature, which is mainly contributed by the presence of four disulfide bridges to produce an overall closed structure. Cyt,c, however, consists of a single polypeptide chain of 104 amino acid residues that are covalently attached to a heme group with an average molecular weight of 12 400.6 The active heme center consists of a porphyrin ring where four pyrrole nitrogens are coordinated to the central Fe atom, forming a square-planar complex. The heme center is surrounded by tightly packed hydrophobic side chains and an outer covering of hydrophilic side groups. Both Lys and Cyt,c are known for providing capping and stabilization to colloidal particles because of their interfacial activities,7 which leads to their conformational changes upon (2) Kovacas, I.; Lundany, A.; Koszegi, T.; Feher, J.; Kovacs, B.; Szolcsanyi, J.; Pinter, E. Neuropeptides 2005, 39, 395. (3) Schenkels, L. C. P. M.; Veerman, E. C. I.; Amerongen, A. V. N. Crit. Rev. Oral Biol. Med. 1995, 6, 161. (4) Yasui, T.; Fukui, K.; Nara, T.; Habata, I.; Meyer, W.; Tsukise, A. Arch. Dermatol. Res. 2007, 299, 393. (5) (a) Thakur, G.; Wang, C.; Leblanc, R. M. Langmuir 2008, 24, 4888. (b) Lu, J. R.; Su, T. J.; Thomas, R. K.; Penfold, J.; Webster, J. J. Chem. Soc., Faraday Trans. 1998, 94, 3279. (c) Lu, J. R.; Su, T. J.; Howlin, B. J. J. Phys. Chem. B 1999, 103, 5903. (6) Harbury, H. A.; Loach, P. A. J. Biol. Chem. 1960, 235, 3640. (7) (a) Haas, A. S.; Pilloud, D. L.; Reddy, K. S.; Babcock, G. T.; Moser, C. C.; Blasie, J. K.; Dutton, P. L. J. Phys. Chem. B 2001, 105, 11351. (b) Wang, L.; Waldeck, D. H. J. Phys. Chem. C 2008, 112, 1351.

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adsorption on water-solid interfaces.8 Our emphasis in the present work is mainly focused on their role of producing metallic (Au) nucleating centers in the absence of any additional reducing agent and their simultaneous adsorption on the surfaces of nanoparticles (NPs), which in turn not only induces conformational changes but also drastically alters the native protein structure.8i Amino acids such as cysteine and glutathione are fine reducing agents, and their presence at appropriate locations in a folded or unfolded protein allows them to be involved effectively in such reactions. Biomaterials synthesized directly by using proteins are far more chemically pure and easy to characterize than by using different reducing and stabilizing agents in the presence of proteins.1h-j Additional reaction reagents not only influence the native state of a protein significantly but also block the active sites of a protein required for nanoparticle complexation. Thus, the synthesis of biomaterials directly involving protein macromolecules for chemical reduction as well as colloidal stabilization helps us to understand the basic mechanism of biomaterial synthesis. To this end, bionanomaterials comprising Au NPs in conjunction with Lys/Cyt,c have been synthesized at 40 and 80 °C to study the influence of folded and unfolded states of protein on the overall shape, structure, and aggregation of NPs. Both Lys and Cyt,c have characteristic UV-visible absorption at around 2855a,9 and 400 nm,10 respectively, due to tryptophan and tyrosine residues of Lys and Π-Π* transitions of the porphyrin rings of Cyc,c. These absorbances are also very sensitive to their conformational changes and denaturation. Likewise, the surface plasmon resonance (SPR) of Au NPs gives a characteristic absorbance at around 520 nm for NPs that are <20 nm,11,12 which is strongly related to the shape and structure of NPs as well as their mode of aggregation. Thus, simultaneous absorbance measurements with respect to reaction time help us to understand the ongoing changes in the physical state of proteins, the kinetics of the NP nucleation process, and the mode of protein-NP association from the very beginning of the reaction. Once bioconjugates of Lys/Cyc,c-Au NPs have been characterized, they are then used in biodegradable protein film formation. Biodegradable protein films have achieved considerable importance because of their uses in food packaging and coating fruits and vegetables and their environmental benefits.13a In addition, they can be used as a film-forming medication (ointment) when high antimicrobial activity against pathogenic microorganisms or infections is required.13b Zein protein films have recently been employed for such purposes because of their low price and ready availability but require considerable (8) (a) Takeda, Y.; Kondow, T.; Mafune, F. J. Phys. Chem. B 2006, 110, 2393. (b) Takeda, Y.; Mafune, F.; Kondow, T. J. Phys. Chem. C 2009, 113, 5027. (c) Bayraktar, H.; You, C. C.; Rotello, V. M.; Knapp, M. J. J. Am. Chem. Soc. 2007, 129, 2732. (d) Aubin-Tam, M. E.; Hamad-Schifferli, K. Langmuir 2005, 21, 12080. (e) Jensen, P. S.; Chi, Q.; Grumsen, F. B.; Abad, J. M.; Horsewell, A.; Schiffrin, D. J.; Ulstrup, J. J. Phys. Chem. C 2007, 111, 6124. (f) Tom, R. T.; Pradeep, T. Langmuir 2005, 21, 11896. (g) Gates, A. T.; Moore, L.; Sylvain, M. R.; Jones, C. M.; Lowry, M.; El Zahab, B.; Robinson, J. W.; Strongin, R. M.; Warner, I. M. Langmuir 2009, 25, 9346. (h) Wallace, J. M.; Dening, B. M.; Eden, K. B.; Stroud, R. M.; Long, J. W.; Rolison, D. R. Langmuir 2004, 20, 9276. (i) Zhang, D; Neumann, O.; Wang, H.; Yuwono, V. M.; Barhoumi, A.; Perham, M.; Hartgerink, J. D.; Wittung-Stafshede, P.; Halas, N. J. Nano Lett. 2009, 9, 666. (9) Yang, T.; Li, Z.; Wang, L.; Guo, C.; Sun, Y. Langmuir 2007, 23, 10533. (10) Jiang, X.; Jiang, J.; Jin, Y.; Wang, E.; Dong, S. Biomacromolecules 2005, 6, 46. (11) (a) Bakshi, M. S. Langmuir 2009, 25, 12697. (b) Bakshi, M. S.; Possmayer, F.; Petersen, N. O. J. Phys. Chem. C 2008, 112, 8259. (c) Bakshi, M. S.; Possmayer, F.; Petersen, N. O. J. Phys. Chem. C 2007, 111, 14113. (d) Bakshi, M. S.; Possmayer, F.; Petersen, N. O. Chem. Mater. 2007, 19, 1257. (e) Bakshi, M. S.; Sachar, S.; Kaur, G.; Bhandari, P.; Kaur, G.; Biesinger, M. C.; Possmayer, F.; Petersen, N. O. Cryst. Growth Des. 2008, 8, 1713. (12) Bakshi, M. S.; Zhao, L.; Smith, R.; Possmayer, F.; Petersen, N. O. Biophys. J. 2008, 94, 855.

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improvement.13a An appropriate blending of such proteins with bioconjugate nanomaterials such as Lys/Cyc,c-Au NPs could improve their workability.

Experimental Section Materials. Chloroauric acid (HAuCl4), lysozyme (Lys) chicken egg white, and cytochrome c (Cyt,c) from bovine heart muscle were purchased from Aldrich. Doubly distilled water was used for all preparations. Synthesis of Au NPs. Aqueous mixtures (10 mL total) of Lys/ Cyt,c (7-80 μM) and HAuCl4 (0.25-1.0 mM) were placed in screw-capped glass bottles. After the components were mixed at room temperature, the reaction mixtures were kept in a waterthermostatted bath (Julabo F25) at precisely 40 or 80 ( 0.1 °C for 6 h under static conditions. The solution changed from colorless to pink-purple and finally to purple within half an hour and remained the same thereafter in most cases. After 6 h, the samples were cooled to room temperature and kept overnight. They were purified from pure water at least two times in order to remove unreacted protein. Purification was done by collecting the Au NPs at 10 000 rpm 5 min after washing each time with distilled water. Methods. UV-visible spectra of as-prepared gold colloidal suspensions were recorded by a UV spectrophotometer (Multiskan Spectrum, model no. 1500) in the wavelength range of 200-900 nm to determine the absorbance due to both protein and the surface plasmon resonance (SPR) of Au NPs. In addition, time-dependent scans of some selective reactions at 40 and 80 ( 1 °C were also carried out to understand the growth kinetics of Au NPs. Atomic force microscopy (AFM) measurements were carried out on a Veeco diCaliber at room temperature. Twenty-five microliters of a purified aqueous colloidal suspension was first spin coated (MTI corporation, PC100) at 500 rpm for 2 min on an ultracleaned glass coverslip and was left to dry in a drybox. The coverslip was then scanned with silicon nitride tips in contact mode to get amplitude and height images. Survey-scanned images were processed and analyzed by using SPM graphic software to obtain the 3D topography of the protein β-sheet bearing Au NPs. Transmission electron microscopy (TEM) measurements were carried out for Au NPs embedded in protein films. Samples were prepared by mounting a drop of a sample on a carbon-coated Cu grid and allowing the sample to dry in air. Each sample was observed with the help of a Philips CM10 transmission electron microscope operating at 100 kV. The pH of some of the reactions was measured with respect to time and temperature. Each reaction solution was placed in a glass container with a double-walled jacket, and the temperature was precisely controlled by externally circulating thermostatted water (Julabo F25). pH measurements were carried out at regular time intervals over a period of 6 h. Protein film casting was carried out by dissolving zein (10% w/v) in aqueous ethanol (90% v/v) along with glycerol (30% on a zein weight basis) as a plasticizer and a 10% Lys/Cyc,c-Au NPs aqueous suspension. This filmogenic solution (5 g) was placed in a 9-cmdiameter plastic Petri dish and gently swirled to coat the bottom of the dish. It was then placed without the lid on a level surface (checked with a spirit level) in an oven at 40 °C for 24 h. The dehydration of this filmogenic solution led to the formation of a protein film with an average thickness of 0.05 mm that was easily peeled off. Films made without glycerol were quite brittle, clear, and less elastic, but the addition of a small quantity of glycerol is required. Color-coordinated data for the films were determined using a Hunter colorimeter model D 25 optical sensor (Hunter Associates). The Hunter L, a, and b color space is a 3D rectangular space based on the opponent color theory. L* is a measure of brightness, and a* and b* are the color coordinates that range (13) (a) Bai, J.; Alleyne, V.; Hagenmeier, R. D.; Mattheis, J. P.; Baldwin, E. Postharvest Biol. Technol. 2003, 28, 259. (b) Singh, N.; Georget, D. M. R.; Belton, P. S.; Barker, S. A. J. Agric. Food Chem. 2009, 57, 4334.

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Bakshi et al. from -90 to þ90 in the color space “circle”. For a*, -90 = green and þ90 = red, and for b*, -90 = blue and þ90 = yellow. To perform the color tolerance, a standard sample is measured and saved for subsequent comparisons. The mechanical properties of the films containing different levels of Lys/Cyc,c conjugate Au NPs were measured using a texture analyzer (TAXT2, Stable Microsystems, Godalming, U.K.) with a 5 kg load cell. The speed was kept constant at 1 mm/min. The well-defined geometry of each film (with average thickness, width, and length of 0.05, 5, and 50 mm, respectively) was used to measure the tensile properties of each film. The measurements were carried out by fixing the ends of a film to two metal plates by using cyanoacrylate glue. It was then single-edge notched to a depth of 2.5 mm midway along the length. Mechanical tests were performed to obtain force/displacement, tensile strength (MPa), and strain at failure data. The tensile strength was measured using σ = Fmax/(w - a)t, where Fmax is the maximum force associated with failure, w is the width of the strip, a is the notch length, and t is the thickness of the strip. Strain at failure was calculated as εfail = Δlmax/l, where Δlmax is the change in the length at Fmax and l is the initial length. At least 10 replicates were tested, and values of the mean and standard deviation were reported.

Results and Discussion UV-Visible Studies. Concentration and Reaction Time Effects. All reactions were simultaneously monitored with UV-visible measurements to determine the course of Au NP synthesis. Some of the typical scans are shown in Figure 1. Figure 1a,b illustrates plots at constant HAuCl4 = 0.25 mM with increasing protein concentration at 80 °C for Lys and Cyc,c, respectively. In both cases, clear absorbance due to Au NPs is visible at around 540 nm at the lowest protein concentration, which diminishes as the amount of protein increases. (See the respective protein absorbances at 270 and 395 nm.) Similar scans are obtained when the reactions were carried out at 40 °C (Supporting Information, Figure S1a,b). The effect of the concentration of gold salt at a constant protein concentration (42 μM) is illustrated in Figure 1c,d. In both cases, an increase in the amount of gold salt from 0.25 to 1.0 mM increases the absorbance due to Au NPs (at ∼540 nm), which means that more NPs are produced at a higher gold salt concentration. It induces a strong influence on the protein absorbances at 270 nm (Figure 1c) and 395 nm (Figure 1d) (indicated by block arrows), which slowly diminishes as the amount of gold salt increases. The progress of Au NP synthesis has also been monitored with time, and typical UV-visible scans are shown in Figure 1e,f at 80 °C for Lys and Cyc,c, respectively. For the Lys reaction (Figure 1e), the absorbance at 270 nm gets more flattened and shows a 20 nm blue shift (inset) with the passage of time, whereas for the Cyc,c reaction (Figure 1f) the intensity at 395 nm decreases8f as the synthesis of Au NPs progresses. This means that the modes of association of Lys and Cyc,c with Au NPs are different from each other. Figure 1f (inset) shows the variation of intensity with time where one can see a close correlation between a decrease in 395 nm and an increase in 540 nm peak intensities and can be related to the progressive association between protein and Au NPs.9,10,14 There is a steep increase in the formation of Au NPs for up to 2 h, which then tends to level off. One can calculate the rate constant for the adsorption of Cyc,c on the Au NP surface by following the linear portion of the 395 nm peak, and it is 1.85  10-5 s-1 for the mole ratios of Au/Cyc,c=6, which is quite comparable to the results in nanoparticle-antibody bioconjugate studies.14b (14) (a) Gomes, I.; Santos, N. C.; Oliveira, L. M. A.; Quintas, A.; Eaton, P.; Pereira, E.; Franco, R. J. Phys. Chem. C 2008, 112, 16340. (b) Soukka, T.; Harma, H.; Paukkunen, J.; Lovgren, T. Anal. Chem. 2001, 73, 2254.

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The above results can be explained on the basis of two factors: temperature-induced denaturation and the strong association between the protein and the NP surface. Both factors work simultaneously. High temperature (80 °C) induces unfolding in both cases, which results in the breaking down of the disulfide bridges exposing reduced cystein residues. The latter initiate the reduction of Au(III) to Au(0), and further nucleation leads to the formation of NPs. A similar reaction occurs at 40 °C, but to a much lesser extent because 40 °C is not expected to induce any significant unfolding in the protein structure. As soon as an NP grows, simultaneous surface adsorption of protein begins because of strong covalent interactions8i between cystein residues and the NP surface and creates a predominantly hydrophobic environment at the protein-NP interface as a result of charge neutralization. The hydrophobic environment encloses tryptophan residues and thus suppresses its absorption at 270 nm in the case of Lys. Similarly, covalent interactions also affect the absorbance due to porphyrin rings in the case of Cyc,c, because the heme is covalently linked to the protein through thioether bridges between the vinyl groups of the heme and the sulfur atoms of two cysteine side-chains. Such an association not only leads to a change in the symmetry15 of heme group but also induces conformation changes, with the absorbance of a Soret band close to 395 nm being almost eliminated. The Q band of Cyc,c usually appears at around 550 nm, but in the present case, it is considered to be overshadowed by the SPR of Au NPs around 540 nm. We will explain further how such an association affects the overall morphology of protein-NPs assemblies later in the discussion section related to microscopic studies. Denaturation and Temperature Effect. A systematic temperature effect in the range of 40-80 °C upon the synthesis of Au NPs has been shown in Figure 1g for a mole ratio of Au/Cyc,c = 31. A gradual increase in the reaction temperature at 5 °C intervals decreases the intensity of the 395 nm peak and increases that of 550 nm, which is quite significant for the former within 40-65 °C and for the latter within 65-80 °C (Figure 1h). Such a marked change in the intensity of the 395 nm peak is not visible in a parallel blank experiment. Figure 1h can be explained in terms of a phase diagram and can be divided into two regions. The first (region I) with a temperature range of 40-65 °C explains the denaturation process of Cyc,c, and the second (region II) with a range of 65-80 °C demonstrates the use of denatured protein in the synthesis of Au NPs. Note that as the denaturation progresses in region I no peak around 550 nm is observed except for the Q band of Cyc,c. However, as soon as the denaturation process is accomplished close to 65 °C, an instantaneous increase in the intensity of the 550 nm peak in region II is observed. This demonstrates that the unfolded state of Cyc,c is more favorable for the reduction of gold ions into nucleating centers and is obviously understood from the greater aqueous exposure of amino acids such as Cys14 and Cys17 as a result of the unfolding of Cyc,c.10 In addition, Cyc,c undergoes more rapid proteolysis in its denatured state than in its native form.16 A significant decrease in the denaturation temperature (Td) of Cyc,c in the absence17 of Au NPs from 85 to 65 °C (in the presence of NPs) is considered to be accompanied by a strong association between the porphyrin ring and the NP surface18 driven by the electron exchange process. (15) Hanrahan, K. L.; Macdonald, S. M.; Roscoe, S. G. Electrochim. Acta 1996, 41, 2469. (16) Wang, L.; Chen, R. X.; Kallenbach, N. R. Proteins: Struct., Funct., Genet. 1998, 30, 435. (17) (a) Jain, R. K.; Hamilton, A. D. Org. Lett. 2000, 2, 1721. (b) Jain, R. K.; Hamilton, A. D. Angew. Chem., Int. Ed. 2002, 41, 641. (c) Wilson, A. J.; Groves, K.; Jain, R. K.; Park, H. S.; Hamilton, A. D. J. Am. Chem. Soc. 2003, 125, 4420. (18) Keating, C. D.; Kovaleski, K. M.; Natan, M. J. J. Phys. Chem. B 1998, 102, 9404.

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Figure 1. (a) Absorbance vs wavelength for colloidal Au NP suspensions prepared at 80 °C and at constant HAuCl4 = 0.25 mM, with varying amounts of Lys. (b) Similar plots with varying amounts of Cyc,c. (c) Plots of Au NP suspensions prepared at 80 °C, constant Lys = 42 μM, and varying amounts of HAuCl4. (d) Similar plots with Cyc,c. (e) UV-visible scans of a Lys þ HAuCl4 þ water ternary reaction (Au/Lys = 6) with time (from 10 min to 6 h) at 80 °C from the beginning of the reaction (lowermost plot) to 6 h (uppermost plot). The inset shows a magnified view of a Lys peak with a blue shift. (f) Similar scans of a reaction with Au/Cyc,c = 6 at 80 °C. The inset shows the opposite variation in the intensities of the Cyc,c peak at 395 nm and the Au NPs peak at 540 nm with time. (g) UV-visible scans of a reaction with Au/Lys=31 from 40 to 80 °C. The inset shows the dependence of the denaturation temperature (Td) on the Au/Cyc,c mole ratio. (h) Variation in the intensity of the Cyc,c peak at 395 nm and the Au NPs peak at 550 nm with temperature. The dotted line shows the Td. See the details in the text.

An increases in the amount of gold salt or an increase in the mole ratio of Au/Cyc,c = 62 and 125 further decreases Td = 57 and 52 °C, respectively. Thus, a regular decrease in the Td (Figure 1g, inset) is clearly related to the increase in the number density of Au NPs. A similar Td for Lys was not possible to determine because of a very small change in 270 nm peak with temperature. Blank Au NPs-Lys/Cyc,c Interactions. A strong association of Au NPs with Lys/Cyc,c can also be understood by simply taking a presynthesized sample of NPs (blank). We synthesized 13538 DOI: 10.1021/la101701f

Au NPs (in the absence of proteins) by reducing HAuCl4 with NaBH4 in the presence of sodium citrate as mentioned in our previous work.11 Figure 2a,b show typical UV-visible scans of blank Au NPs with increasing amounts of Lys and Cyc,c, respectively. The dotted-line scan in each case belongs to blank Au NPs in the absence of protein with clear absorbance at around 520 nm. The intensity of this absorbance shows a significant increase with a red shift of 40 nm as soon as a small amount of protein is added in both cases. The increase in the intensity as well Langmuir 2010, 26(16), 13535–13544

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Figure 2. (a) UV-visible scans of a reaction with a constant amount of 0.25 mM HAuCl4 and varying amounts of Lys from 1 (top plot) to 10 μM (bottom plot) at 80 °C. (b) Similar UV-visible scans of a reaction with Cyc,c. (c) Intensity plots of an Au NP peak at 550 nm vs Lys concentration at 40 and 80 °C. (d) Similar plots with Cyc,c. (e) Variation in the reaction pH with time for different Au/Cyc,c mole ratios at 40 °C. (f) Plot of limiting pH values with protein concentration at 40 and 80 °C for both proteins. See the details in the text.

as a red shift in the absorbance occurs because of self-aggregation among the NPs induced by Lys/Cyc,c. The addition of Lys (Figure 2a) dramatically suppresses the SPR of Au NPs8i (solid arrow) with the clear emergence of a Lys peak at around 270 nm (dotted arrow). A decrease in the intensity with the amount of Lys is plotted in Figure 2c, with a predominant effect at 80 °C rather than at 40 °C. From the break in the plot at 80 °C, it is possible to determine the maximum amount of Lys (i.e., 0.0055 mM) complexed with Au NPs that gives a Au/Lys mole ratio of 91. This value is about 3 times that of the minimum amount of Lys used for the same amount of gold salt (i.e., Au/Lys = 31, see Figure 1a) and suggests that only one-third the amount of Lys is complexed with blank NPs in comparison to in situ complexation during the reduction reaction. Thus, the simultaneous reduction of gold ions into nucleating centers will have a greater probability of synergistic interactions between Lys and NPs at a microscopic level than by using presynthesized NPs. The Au nucleating centers produced upon the reduction of AuCl4- ions are expected to have a greater potential to break the disulfide groups and form covalent bonds8i in comparison to much larger NPs. However, Langmuir 2010, 26(16), 13535–13544

no change in the magnitude of intensity is observed for Cyc,c (Figure 2d) except a red shift of 40 nm, which means that the mode of aggregation is different in both cases. NPs embedded deeply and covered with several protein layers will not be very SPRactive in comparison to those simply associated with protein polyelectrolytes. Thus, Figure 2a might represent the former type of complexation, and Figure 2b can be related to the latter. This will be further discussed with AFM and TEM studies. Influence of pH. The pH of each reaction without using any buffer remains fairly constant and is acidic in nature because of the dissociation of HAuCl4. Figure 2e shows typical pH plots of a few reactions in the presence of Cyc,c. Similar behavior is observed for reactions in the presence of Lys (Figure S2). The pH value in each case decreases slightly within 30 min of the reaction and then remains fairly constant. A variation in the limiting pH values at 40 and 80 °C with protein concentration has been shown in Figure 2f. It increases with the increase in the concentration of protein with a predominant effect in the case of Cyc,c compared to that for Lys. The structure of Cyc,c in the presence of ionic residues on its periphery is considered to be DOI: 10.1021/la101701f

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Figure 3. AFM height images of different samples prepared with mole ratios of (a) Au/Lys = 31 (scan range of 5 μm), (b) Au/Lys = 3 (scan range of 3.6 μm), and (c) Au/Lys = 62 (scan range of 4 μm) at 80 °C. (d-f) TEM images of these samples. See the details in the text.

a more open structure than Lys and hence will have a greater affinity for protonation as the amount of Cyc,c increases. In addition, the isoelectric points (Ip’s) of Lys and Cyc,c are quite close to each other and are at a much higher pH (∼10 to 11); therefore, they will have a drastic effect on the protein native state at low pH (∼3 to 4). A breakdown in tertiary structure at low pH is expected to facilitate the reduction of gold ions by cystein groups. If the isoelectric points (Ip’s) of Lys and Cyc,c (pH ∼10 to 11) are maintained in the reaction, then a significant number of Au NPs are not produced over a period of 6 h at 40 and 80 °C (Figure S3a,b). Thus, the positively charged nature of protein (at low pH, Figure 2e) is essential to the synthesis of Au NPs because it helps in the electrostatic interactions with AuCl4- ions that initiate the reduction reaction. Microscopy Studies. Lysozyme-Au NPs. The mode of association between Cyc,c/Lys and Au NPs in the form of bioconjugate nanomaterials can be best understood from the imaging studies. Figure 3 shows AFM images of different samples with varying amounts of Lys. A height image (Figure 3a) of a sample with a mole ratio of Au/Lys = 31 prepared at 80 °C shows large groups of Lys conjugate NPs. The size of a group of NPs can be determined from the line analysis and is 423 ( 35 nm with a height of 173 ( 23 nm. An increase in the amount of Lys with a mole ratio Au/Lys = 3 (Figure 3b) produces similar morphologies with a smaller size of 277 ( 17 nm and a height of 133 ( 15 nm. Both images also show that most of the aggregates (in low contrast) are embedded deep in the denatured Lys layers and become clear as the amount of Lys is decreased (Figure 3c) with size 268 ( 19 nm and a height of 113 ( 18 nm. The corresponding 13540 DOI: 10.1021/la101701f

TEM images of the samples are shown in Figure 3d-f. Figure 3d shows a number of large flakes entrapping Au NPs, and their size is quite comparable to that of groups of Lys conjugate NPs shown in Figure 3a. Figure 3e shows a few flakes at higher magnification with a greater amount of Lys (Au/Lys = 3). Flakes are formed when unfolded protein at high temperature (i.e., 80 °C) undergoes dehydration, which in turn self-associates because of predominant hydrophobic interactions into a gel state.19 This process is considered to entrap NPs of 2 to 3 nm that are already capped and stabilized by protein macromolecules. The nature of the gel state varies with the amount of Lys used. In Figure 3e, it is flakelike, but a decrease in the amount of Lys by 20-fold with Au/Lys = 62 does not show any flake formation (Figure 3f). Self-aggregated Lysentrapping Au NPs are clearly visible in Figure 3f. The samples prepared at 40 °C do not show such morphologies. Singly dispersed large NPs are present in these samples (Figure 4). Figure 4a shows the AFM image of a sample with Au/Lys = 31, and Figure 4b,c shows the AFM image of a sample with Au/Lys = 62. Figure 4a,d shows complementary AFM and TEM images. Several Au NPs of 15 ( 7 nm (see size distribution histogram in Figure 4e) are entrapped in the protein film but in no way look flakelike. Figure 4f also shows a similar physical appearance at Au/Lys = 62 with double the amount of gold salt. AFM images in Figure 4b,c do not clearly supplement this information, which might be due to the presence of large aggregates with a greater (19) (a) Beveridge, T.; Jones, L.; Tung, M. A. J. Agric. Food Chem. 1984, 32, 307. (b) Kanno, C.; Mu, T. H.; Hagiwara, T.; Ametani, M.; Azuma, N. J. Agric. Food Chem. 1998, 46, 417.

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Figure 4. AFM height images of different samples prepared with mole ratios of (a) Au/Lys = 31 (scan range of 1.5 μm) and (b, c) Au/Lys = 62 (scan range of 1.75 μm) at 40 °C. Image b is a lateral resolution image of c. (d, f) Corresponding TEM images of these samples. (e) Size distribution histogram of image d. See the details in the text.

number of Au NPs. The absence of gel-state or flake like morphologies is simply due to the low reaction temperature of 40 °C, which causes less dehydration19a than a reaction at 80 °C. In addition, the presence of larger NPs at 40 °C (15 ( 7 nm) than at 80 °C (2 to 3 nm) is simply related to the reduction efficiency of Lys. At 80 °C, the unfolded state of Lys allows the maximum reducing potential of cystein amino acids to be used in converting the gold ions into nucleating centers compared to a relatively folded state of Lys at 40 °C. Stronger reducing conditions always produce a greater number of nucleating centers, which in turn produce smaller NPs than are produced by a smaller number of nucleating centers.11 Cytochrome,c-Au NPs. The results for the samples prepared in the presence of Cyc,c are presented in Figures 5 and 6. These images show a significant difference between the nature of Cyc,cAu NPs interactions from that of Lys-Au NPs. Here, the Cyc,cAu NPs aggregates assemble in a very ordered fashion. Figure 5a, b shows long strands of such aggregates produced from Au/ Cyc,c = 31 at 80 °C. However, a close-up image (Figure 5c) of a sample with Au/Cyc,c = 62 indicates the presence of polyhedral aggregates with an average size 56 ( 7.8 nm determined from line analysis. TEM images give further insight into the morphology of such aggregates with better resolution. Figure 5d indicates (via dotted circles) the presence of several interconnected 48 ( 8 nm spherical micelles of Cyc,c bearing small Au NPs. Note that the size determined from the line analysis in Figure 5c is slightly overestimated. A magnified view of a single micelle loaded with several NPs of 2 to 3 nm is shown in Figure 5e. An increase in the amount of Cyc,c with Au/Cyc,c = 6 predominantly deforms the spherical shape, and micelles exist in a rather more fused state (Figure 5f). Surprisingly, if the same reaction is carried out at 40 °C then no micelle formation is observed; instead long, clear strands of aggregates are observed for a sample with Au/Cyc,c = 31 (Figure 6a). Each aggregate is made of a few large Au NPs wrapped in Cyc,c. A close-up image further clarifies this in Figure 6b, where one can even estimate the size of Cyc,c conjugate Langmuir 2010, 26(16), 13535–13544

NPs of 58 ( 21 nm by following the peaks and valleys (Figure 6c) in a single strand. Complementary TEM images further confirm this. Figure 6d shows a linear arrangement of large NPs of 35 ( 24 nm (histogram in inset) in a single strand driven by capped Cyc,c. Note two filled block arrows pointing to two Au NPs connected through a bridge of Cyc,c indicated by an empty block arrow. The high-resolution image of this portion is shown in Figure 6e with a connecting bridge as dotted lines. Figure 6e,f also shows that Cyc,c not only drives them in the form of long strands but also caps them in the form of small aggregates. That is why the size evaluated from the line analysis (Figure 6c) is larger than that determined from TEM images. A comparison between the morphologies of the aggregates of Figures 5 and 6 illustrates a clear difference between the nature of the same reaction carried out at different temperatures. Though in both places a simple reduction of gold ions into atoms has taken place, a marked difference in the physical state of Cyc,c induced a significant difference not only in the overall shape and structures of Au NPs but also in their mode of aggregation. Relative Comparison between Lys and Cyc,c. Microscopy studies explain a contrasting difference between the modes of association of Lys and Cyc,c with Au NPs. Lys exists in the form of liquid-crystalline flakes at 80 °C and a typical gel at 40 °C enclosing NPs, whereas Cyc,c forms clear spherical micelles at 80 °C and bioconjugate protein-NP fibrils at 40 °C. As mentioned earlier in the UV-Visible Studies section, the reduction is primarily achieved by the cystein residues in both cases, and as soon as the Au nucleating centers are created, they simultaneously associate with protein. (See a schematic representation of the reaction mechanism in Figure 7.) At 40 °C, the protein is considered to be mainly in the folded state, but the protein-NP association further induces unfolding because of a partial breakdown of some of the disulfide bonds (step a in Figure 7). The unfolded protein then interacts with another protein (step b) to induce selfaggregation, bringing about several protein-conjugated NPs in DOI: 10.1021/la101701f

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Figure 5. AFM height images of different samples prepared with mole ratios of (a, b) Au/Cyc,c = 31 (scan range of 5.39 μm) and (c) Au/ Cyc,c = 62 (scan range of 2 μm) at 80 °C. (d) Corresponding TEM image of sample a, with image e showing only a single Cyc,c micelle loaded with Au NPs. (f) TEM image of a deformed or fused micelle of the sample prepared with Au/Cyc,c = 6 at 80 °C. See the details in the text.

Figure 6. (a, b) AFM height images of a sample prepared with a mole ratio of Au/Cyc,c = 31 (scan range of 2.2 μm) at 40 °C. (c) Line analysis of (b). (d-f) TEM images of the same sample showing a chainlike arrangement of NPs along with Cyc,c. The inset in d is the size distribution histogram of Au NPs. See the details in the text.

a self-assembled state8i in the case of Lys. This is not the case with Cyc,c, which is even smaller than Lys. Cyc,c is known for fibril formation20 (step d in Figure 7), a typical kind of self-assembled state, especially for the small proteins,21 where polypeptide (20) (a) de Groot, N. S.; Ventura, S. Spectroscopy 2005, 19, 199. (b) Pertinhez, T. A.; Bouchard, M.; Tomlinson, E. J.; Wain, R.; Ferguson, S. J.; Dobson, C. M.; Smith, L. J. FEBS Lett. 2001, 495, 184. (21) (a) Jarrett, J. T.; Lansbury, P. T. Biochemistry 1992, 31, 12345. (b) Devlin, G. L.; Knowles, T. P. J.; Squires, A.; McCammon, M. G.; Gras, S. L.; Nilsson, M. R.; Robinson, C. V.; Dobson, C. M.; MacPhee, C. E. J. Mol. Biol. 2006, 360, 497.

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strands composed of antiparallel β-sheets in a cross-β arrangement exist. It happens when the native folds of highly R-helical protein are destabilized by Cyc,c adsorption on an NP surface (step e) and subsequently interact with other free or conjugated proteins. At 80 °C, extensive dehydration (step c) turns the self-assembled Lys-Au NPs into large liquid-crystalline flakes because of its relatively less hydrophilic nature compared to that of Cyc,c. However, it is difficult to determine the isotropic-nematic phase-transition behavior of such a complex system; nevertheless, several reports indicate that predominantly hydrophobic proteins Langmuir 2010, 26(16), 13535–13544

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Figure 7. Schematic representation of the proposed reaction mechanism for the synthesis of Lys/Cyc,c conjugated Au NPs. See the details in the text.

do exist in the form of flakes.22 However, long Cyc,c-Au NPs strands (step f) convert into typical micelles at 80 °C. Temperature-induced micelle formation of Cyc,c23 is quite similar to that of amphiphilic triblock polymers.24 Because Cyc,c is roughly spherical in shape and its surface bears several cationic and anionic residues, the unfolding of Cyc,c thus leads to an open structure with a greater probability of hydrophobic domains coming into contact with the aqueous phase. To avoid this, self-aggregation leading to roughly spherical micelle formation sets in, where different Cyc,c macromolecules self-assemble to enclose the hydrophobic domains in the interior of the micelle and ionic residues remain in contact with aqueous phase. Because it occurs only when Cyc,c is in its fully unfolded state, we do not see any micelle formation or micelles with Au NPs at 40 °C. Protein Film Formation and Properties. The above characterized samples of Lys/Cyc,c-Au NPs bioconjugates have been further used in biodegradable protein film formation. Because of the high water solubility of pure Lys and Cyc,c, as well as the colloidal nature of Lys/Cyc,c-Au NPs bioconjugates, it is not possible to use them directly in protein film formation for a range of applications under different conditions. However, a waterinsoluble protein such as zein can be used in conjunction with the present bioconjugate nanomaterials to develop protein films with better mechanical properties (Introduction). For this purpose, two samples with Au/(Lys or Cyc,c) = 6 and 12 have been selected for protein film formation (Experimental Section), and the resulting films are shown in Figure 8. All films are semitransparent and absorb in the UV region (Figure S4). The degree of scattering is too high for a pure zein film but decreases along with pure Lys/Cyc,c, and completely vanishes in the presence of (22) (a) Mizuno, A.; Mitsuiki, M.; Motoki, M. J. Agric. Food Chem. 2000, 48, 3286. (b) Mizuno, A.; Mitsuiki, M.; Motoki, M.; Ebisawa, K.; Suzuki, E. i. J. Agric. Food Chem. 2000, 48, 3292. (c) Inouye, K.; Nakano, K.; Asaoka, K.; Yasukawa, K. J. Agric. Food Chem. 2008, 57, 717. (23) Brault, P. A.; Kariapper, M. S. T.; Pham, C. V.; Flowers, R. A.; Gunning, W. T.; Shah, P.; Funk, M. O. Biomacromolecules 2002, 3, 649. (24) (a) Bakshi, M. S.; Bhandari, P. J. Photochem. Photobiol., A 2007, 186, 166. (b) Bakshi, M. S.; Kaur, N.; Mahajan, R. K. J. Photochem. Photobiol., A 2007, 186, 349. (c) Bakshi, M. S.; Kaur, N.; Mahajan, R. K. J. Photochem. Photobiol., A 2006, 183, 146. (d) Bakshi, M. S.; Sachar, S. J. Colloid Interface Sci. 2006, 296, 309. (e) Bakshi, M. S.; Sachar, S.; Singh, K.; Shaheen, A. J. Colloid Interface Sci. 2005, 286, 369. (f) Bakshi, M. S.; Sachar, S.; Yoshimura, T.; Esumi, K. J. Colloid Interface Sci. 2004, 278, 224.

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bioconjugate NPs to produce more isotropic films. The colorimeteric measurements also help us to determine visible spectroscopic effects on the basis of color contrast. L*, a*, and b* values (Experimental Section) of zein films containing pure Lys, and bioconjugate NPs (of Au/Lys = 6 and 12) are shown in Figure 8e. The films containing Au NPs have lower L* and b* values and less-negative a* values in comparison to a film without NPs. A lower L* value indicates that the film is less bright or more dark with the inclusion of NPs, which is obviously expected on the basis of SPR of NPs. Likewise, a* becomes less negative with a shift from green toward the red zone as a result of enhanced SPR, which absorbs at around 520 nm in the visible spectrum. The variation in b* is considered to be entirely related to the protein structure because it refers to the blue and yellow parts of the electromagnetic spectrum. The positive b* value indicates a light-yellow color in the protein film that decreases in the presence of NPs. The dried state of a protein film is in fact a denatured form of protein polyelectrolytes where charge neutralization among the dipolar amino acids promotes its hydrophobic nature. The presence of NPs reduces it to some extent because the adsorption of protein onto the NP surface is primarily a electrostatically driven process with the result being that the b* value decreases. A similar trend (Figure S5) in all parameters is observed when Cyc,c-Au NPs are used instead of Lys-Au NPs in the protein films. The photophysical properties of the films are further correlated with the mechanical properties. Figure 8f shows the tensile strength of the protein films made from both Cyc,c-Au NPs and Lys-Au NPs. In both cases, the trend is identical to the maximum tensile strength of the film in the absence of NPs that decreases with the increase in Au/protein from 6 to 12. This is expected on the basis of the incorporation of NPs in the tertiary protein structure whereby they decrease the cohesive interactions by breaking the disulfide groups. Better isotropic effects (as evident from the UV studies) and lower tensile strength make the protein film more brittle (see Figure S6 for strain at failure) for better application in devices where they can withstand even greater temperature variations (% weight loss in Figure S7). Figure 8f also suggests that the tensile strength is greater for film made from Cyc,cAu NPs rather than Lys-Au NPs. Because the tensile strength is an intensive property of the film, it depends on the composition of DOI: 10.1021/la101701f

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Figure 8. Photographs of protein films made from (a) zein þ [Au NPs/Lys = 6], (b) zein þ [Au NPs/Lys = 12], (c) zein þ [Au NPs/Cyc,c = 6], and (d) zein þ [Au NPs/Cyc,c = 12]. See the Experimental Section for the composition of different components. (e) Histogram of color coordinates for the films made from zein þ [Au NPs/Lys]. (f) Histogram of the tensile strength of the same films. 1, 2, and 3 stand for [Au NPs/ Lys] = 0, 6, and 12, respectively. See the details in the text.

the film. A film made with long strands of Cyc,c-conjugated NPs (Figure 6d) can easily allow them to assimilate into the denatured liquid-crystalline phase rather than into self-assembled large aggregates of Lys-Au NPs (Figure 4d,f). Therefore, the basic arrangement of NPs in the bioconjugate nanomaterials is the key factor in determining the mechanical properties and their appropriate applications.

Concluding Remarks This work is related to the synthesis of bioconjugate nanomaterials by directly using simple, low-molecular-weight, water-soluble, well-characterized important proteins such as Lys and Cyc,c as weak reducing and stabilizing agents under different experimental conditions. Low-temperature (40 °C, predominantly the native state of protein) synthesis produces Au NPs of >10 nm dimensions arranged in the form of long strands in the presence of Cyc,c, and in a self-aggregated state in the case of Lys. But high-temperature (80 °C, unfolded state of protein) synthesis leads to the formation of Cyc,c 13544 DOI: 10.1021/la101701f

micelles loaded with small NPs of 2 to 3 nm and flakes of Lys entrapping such NPs. Thus, both proteins produce similar shapes and sizes of NPs under different conditions, but the arrangement of NPs in bioconjugate materials is much different and is simply explained on the basis of seeding growth triggered by the unfolded Lys in conjunction with Au NPs. Similar growth in the case of a much smaller protein such as Cyc,c leads to fibril formation along with Au NPs, which ultimately appeared in the form of long strands. The appropriate use of such bioconjugate nanomaterials in protein film formation paves the way to controlling the photophysical and mechanical properties of such films for various biomedical applications. Acknowledgment. These studies were partially supported by financial assistance from CSIR (ref. no. 01(2220)/08/EMR-II), New Delhi, India. Supporting Information Available: UV-visible spectra and film properties. This material is available free of charge via the Internet at http://pubs.acs.org. Langmuir 2010, 26(16), 13535–13544